The present invention relates to the capture of cells or other material from a sample suspension and the deposition of cells or other material onto an appropriate receiving surface, such as a microscope slide for observation.
In the field of cytology, human and machine vision systems perform effectively on near mono-layer depositions of cellular material. Expanded use of machine vision for slide examination, a growing number of special stains and the development of molecular markers have increased the need to prepare multiple representative depositions or multiple slides from the same sample suspension. However, care must be taken in the deposition of cellular material used for cytological examination. In particular, to diagnose disease, slides must be representative of the sample suspension, which ideally is representative of the patient. And if multiple cellular depositions are made on the same or on different slides, then each of these depositions must also be representative of the sample suspension. Achieving an appropriate concentration and distribution of material for examination or analysis is a limitation of many sample preparation techniques. Therefore an intent of the present invention is to overcome some of these limitations.
Three common techniques used to deposit cells from a sample suspension onto microscope slides are: centrifugation, filter transfer and fluid evaporation. Examples of centrifugation are taught in U.S. Pat. No. 4,391,710 to Gordon entitled “Cytocentrifuge”, U.S. Pat. No. 5,679,154 to Kelley et al. entitled “Cytology centrifuge apparatus ”, U.S. Pat. No. 5,480,484 to Kelley et al. entitled “Cytology centrifuge apparatus”, U.S. Pat. No. 6,162,401 to Callaghan, entitled “Cytofunnel arrangement”, and U.S. Pat. No. 5,419,279 to Carrico, Jr. et al. entitled “Apparatus for depositing and staining cytological material on a microscope slide”.
Filter transfer is taught in U.S. Pat. No. 4,395,493 to Zahniser and U.S. Pat. No. 5,976,824 to Gordon entitled “Method and apparatus for collecting a cell sample from a liquid specimen”. And an example of fluid evaporation is taught in U.S. Pat. No. 5,784,193 to Ferguson entitled “Microscope slide with removable layer and method”.
Variations of these methods are taught in U.S. Pat. No. 5,419,279 to Carrico, Jr. et al. entitled “Apparatus for depositing and staining cytological material on a microscope slide”, U.S. Pat. No. 6,225,125 to Lapidus, entitled “Method and apparatus for controlled instrumentation of particles with a filter device”, U.S. Pat. No. 6,309,362 to Guirguis entitled “Method and apparatus for automatically separating particulate matter from a fluid”, and U.S. Pat. No. 6,358,474 to Dobler et al. entitled “Device and Method for Isolating Cell Material Out of a Tissue Medium and/or a Liquid”.
For filter transfer, cellular or other material is collected, typically on a circular filter, and is transferred to the microscope slide by contact, back-pressure or a combination of contact and back-pressure. Other examples of cell deposition onto membrane filters in the prior art are taught by FIG. 4 of U.S. Pat. No. 5,419,279 to Carrico Jr. et al.; FIG. 11 in U.S. Pat. No. 5,679,154 to Kelley et al.; FIG. 2 of U.S. Pat. No. 4,250,830 to Leif; FIG. 3 in U.S. Pat. No. 6,162,401 to Callaghan; and FIG. 5 of U.S. Pat. No. 6,309,362 to Guirguis.
U.S. Pat. No. 6,162,401 to Callaghan teaches cell capture on a filter or membrane in which the filter dimensions are smaller than that of a microscope slide. This prior art does not teach or derive advantage by capturing material on a filter which extends beyond the dimensions of the receiving surface. While capturing material on filters, filter dimensions are typically kept to a minimum since filter deformation may cause inconsistencies in flow and thus material capture by the filter. Under less favorable conditions the filter itself could tear, otherwise fail or its characteristics may be compromised. Similarly, although filters are often supplied in a support structure, when material distribution is important for analysis, flow impediments in the vicinity of the carrier or support structure are viewed as problematic. Therefore, filter dimensions are generally kept to a minimum. Typically, the filter area is smaller and fits entirely within the dimensions of the receiving surface. An example of a departure from this in the prior art is U.S. Pat. No. 5,784,193 to Ferguson, which maintains its advantages and exploits situations when material dimensions exceed that of the exposed region of the slide or receiving surface.
Currently, in cytology, if multiple slides are required from a sample suspension, either the sample is split prior to deposition, or multiple portions (sub-samples) are captured on individual filters, and these are then deposited onto one or more slides for analysis. Multiple depositions are taught in: U.S. Pat. No. 4,250,830 to Leif entitled “Swinging buckets”, U.S. Pat. No. 4,961,432 to Guirguis, entitled “Modular fluid sample preparation assembly”, and U.S. Pat. No. 5,784,193 to Ferguson, entitled “Microscope slide with removable layer and method”. The latter reference is of particular interest since it teaches: precise confinement of material to region(s) of interest; protecting the slide from contamination during bulk processing; and independent staining of various regions on the same slide. Products manufactured under this patent include high-tech surface coatings of PVC type materials that are easily removed, resistant to abrasion, and stable during cell fixation and staining. Such coatings as applied in fluid or vapor state are referred to as evaporation methods (see Ferguson column 6, lines 24–29). Additionally, Ferguson specifically teaches the limitations placed on the examination of cellular material when cells are deposited near the edge of the coverslip or microscope slide.
One limitation of using multiple small filters to capture multiple portions of material is that flow rate and other conditions for cell capture must be monitored closely to prevent non-representative samples or inadequate preparations. A non-representative sample, for example, may lack cancerous cells from which to make a diagnosis. Similarly, excess material, sparseness or substantial variations in cellular concentration may impede or otherwise confound diagnosis.
Some of these filter limitations are taught in U.S. Pat. No. 4,395,493 to Zahniser entitled “Monolayer device using filter techniques”, wherein the capture of cellular material on a filter tape is monitored by a cell counter. U.S. Pat. No. 4,614,109 to Hoffman teaches membrane testing by measuring differential pressure across it. U.S. Pat. No. 6,010,909 to Lapidus and U.S. Pat. No. 6,225,125 to Lapidus teach blocking pores. As material is captured, membrane pores are blocked, thus the differential pressure across the membrane provides an estimate of material concentration collected on the filter prior to deposition on a receiving surface. These techniques are designed to ensure that the appropriate concentration of material is captured on each filter. In order to achieve the appropriate concentration of material, however, these techniques are sometimes complicated and require expensive equipment and a substantial amount of time to perform.
Although the concentration of the collected material can be monitored, one difficulty with using multiple filters results not from the design of the device for monitoring the concentration, but from the nature of biological samples. Even in homogeneous samples, various sized clumps of cells, mucus, debris, particulate matter and various contaminants may be present. Therefore, that material which is captured on one small filter may be substantially different than the material captured on a subsequent filter. For a relatively large deposition of material, a few cell clumps or inadequate areas are not uncommon and may or may not impede diagnosis. The probability of capturing non-representative material is related to the surface area of the filter on which the material is collected.
A related difficulty is that once any material is removed from the sample suspension, the characteristics of the sample have changed and replicates are no longer possible. Unfortunately, with filter transfer methods, as cells are captured on the membrane, the concentration of constituents in the sample suspension are altered and therefore subsequent preparations from this sample suspension may no longer be representative. And in some cases, once any material has been removed from the original sample suspension, additional preparations from this may not even be suitable for the intended use. Similarly, repeated blots from the same area of a filter will not produce representative slides.
Another limitation for many analysis techniques, including cytology, is that to be effective, the concentration of material must fall within a target range. Still other test protocols require a target range of specific sample constituents. These target ranges are used to exploit malignancy-associated changes, for example which require predominantly DNA stained, non-overlapping nuclei. Typically, preparations for exploiting malignancy-associated changes include scrapings, aspirates, and washings for the detection of cancer and other diseases. Some of these applications are taught in U.S. Pat. No. 5,889,881 to MacAulay et al. and U.S. Pat. No. 6,026,174 to Palcic et al.
While most cytology-based tests simply require representative samples containing abnormal cells, malignancy-associated changes are measured on ostensibly normal cells. Unfortunately, in the majority of cases the concentration of cells and constituents, in any given sample, is not known a priori. While cell counters, sample dilutions, differential pressure and other techniques are commonly employed to monitor or otherwise control the concentration of cells deposited, these require additional equipment, time and expertise. Even then for a variety of reasons the resulting cell deposition may be inadequate. It is therefore a goal of the present invention to improve the probability that an area adequate for analysis will be deposited on the receiving surface.
The need exists for a rapid, simple, cell deposition method to prepare multiple representative slides from a sample suspension. In addition, a more restricted set of applications would benefit from a cell or material deposition in the form of a concentration gradient.